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Industrial Crops & Products

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Elevating fermentation yield of cellulosic lactic acid in calcium lactate formfrom corn stover feedstock

Chengxiang Weia,1, Gang Liua,1, Jian Zhanga,b,⁎, Jie Baoa,⁎

a State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, Chinab Key Laboratory for Solid Waste Management and Environment Safety (Tsinghua University), Ministry of Education of China, Tsinghua University, Beijing, 10084, China

A R T I C L E I N F O

Keywords:Chiral lactic acidLignocelluloseCalcium lactateSodium lactateSimultaneous saccharification and co-fermentation (SSCF)

A B S T R A C T

Chiral lactic acid is recovered as sodium lactate or calcium lactate from fermentation broth, then the lactate arereleased to its lactic acid form as monomer chemical of polylactic acid (PLA). This study investigated the fer-mentation behaviors of chiral lactic acid from corn stover feedstock when lactic acid was recovered in differentlactate forms, calcium lactate or sodium lactate. When chiral lactic acid is recovered in calcium lactate form inthe simultaneous saccharification and co-fermentation (SSCF) of corn stover feedstock, L-lactic acid titer reached130.3 g/L, approximately 14% greater than that in sodium L-lactate form; D-lactic acid titer reached 124.8 g/L,approximately 11.2% greater than that in sodium D-lactate form. The lessened lactate inhibition in calciumlactate form on cellulase activity and cell viability was found to be responsible for the elevated cellulosic L- andD-lactic acid yield. The techno-economic analysis showed the greater economic advantage of chiral lactic acid incalcium lactate than that in sodium lactate. This study provides the strong evidence and simple option forimproving cellulosic chiral lactic acid yield.

1. Introduction

Lignocellulose is the only promising feedstock for production ofchiral lactic acid as the monomer chemical of biodegradable polylacticacid (PLA) (Liu et al., 2015; Werpy et al., 2004). During the fermen-tative production from lignocellulose feedstock, the alkaline agent isadded to neutralize the lactic acid generated to keep a suitable pHenvironment for lactic acid bacterium strains (Liu et al., 2015; Yi et al.,2016; Zhao et al., 2013). The neutralization agents are either sodiumhydroxide to recover the lactic acid in the form of sodium lactate, orcalcium hydroxide to recover the lactic acid in the form of calciumlactate. To obtain the chiral lactic acid with high purity, the calciumlactate or sodium lactate have to be recovered by either adding sulfuricacid to release lactic acid from calcium lactate, or electrodialysis torelease lactic acid from sodium lactate (Wasewar, 2005). The first op-tion to release lactic acid from calcium lactate is seemingly the pre-ferred choice for its better feasibility and the lower cost on generatingcalcium sulfate precipitate (Chen et al., 2012; Frieling and Schugerl,1999). However, the previous studies generally took the sodium lactateform as the product using NaOH as the neutralizing agent, instead ofthe operation simplicity of Ca(OH)2 solution (Qureshi et al., 2017;

Zhang et al., 2016).This study evaluated the cellulosic lactic acid fermentation using

both Ca(OH)2 and NaOH as the neutralizing agents to generate calciumlactate and sodium lactate in the high solids loading simultaneoussaccharification and co-fermentation (SSCF) of pretreated and biode-toxified corn stover feedstock. Chiral pure L- and D-lactic acid fer-mentation by two genetically modified Pediococcus acidilactici strainsfitting for co-fermentation of glucose and xylose was tested. A pre-liminary economic analysis was conducted to evaluate the feasibility ofusing Ca(OH)2 to replace NaOH as the neutralizing agents during lacticacid production.

2. Materials and methods

2.1. Raw materials and enzymes

Corn stover was obtained in fall 2016 from Tongliao, InnerMongolia, China. The dried corn stover was milled to pass through a10mm diameter screen in the beater pulverizer and stored in plasticbags at room temperature before use. The raw corn stover contained33.0% glucan, 26.9% xylan, 20.8% lignin, and 6.3% ash on a dry

https://doi.org/10.1016/j.indcrop.2018.10.041Received 27 June 2018; Received in revised form 25 September 2018; Accepted 13 October 2018

⁎ Corresponding authors at: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai,200237, China.

1 These authors contributed equally to this work.E-mail addresses: jzhang@ecust.edu.cn (J. Zhang), jbao@ecust.edu.cn (J. Bao).

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weight basis analyzed using the two-step acid hydrolysis method ac-cording to National Renewable Energy Laboratory (NREL) protocols(Sluiter et al., 2012).

The cellulase enzyme used in this work was Cellic CTec2(Novozymes, Tianjin, China, batch No.VCS10008). The filter paperactivity of Cellic CTec2 was 203.2 FPU/mL determined using the NRELprotocol LAP-006 (Adney and Baker, 1996), and the cellobiase activitywas 4900 CBU/mL determined according to Ghose (1987). The proteincontent of cellulase was 87.3 mg/mL determined by the Bradford(1976) method using bovine serum albumin (BSA) as protein standard.

2.2. Strains and cultivation method

Amorphotheca resinae ZN1 (CGMCC 7452, China General MicrobialCollection Center, Beijing, China) was used as the biodetoxificationstrain for degrading inhibitors existing in the pretreated corn stover viasolid state fermentation (Zhang et al., 2010a).

P. acidilactici TY112 (CGMCC 8664), P. acidilactici ZY271 (CGMCC13611), and P. acidilactici ZY15 (CGMCC 13612) were cultured in thesimplified MRS medium used for cultivating the seeds (Qiu et al., 2017,2018; Zhao et al., 2013).

2.3. Pretreatment and biodetoxification

Corn stover was pretreated using the dry acid pretreatment methodaccording to Zhang et al. (2011) and He et al. (2014). Briefly, the driedcorn stover and a dilute sulfuric acid solution at 7.2% (w/w) were

concurrently fed into a reactor at a solid/liquid ratio of 2:1 (w/w) at175 °C for 5min under mild helical agitation to yield the pretreatedcorn stover with dry solids content of about 50% (w/w). No pretreat-ment liquid generated during pretreatment.

The pretreated corn stover was biodetoxified by A. resinae ZN1 ac-cording to Zhang et al. (2010a) and He et al. (2016). Briefly, the sulfuricacid in the pretreated corn stover was neutralized by Ca(OH)2 to pH5.5, then disk milled to remove the extra-large fibers before inoculatingthe A. resinae ZN1 seeds at 10% (w/w) ratio in a 15 L bioreactor. Thebiodetoxification was lasted for 3 days at 1.0 vvm of aeration and aslight periodical agitation (at 50 rpm for 30 s) every 12 h. No additionalfresh water or nutrients were supplemented.

2.4. Simultaneous saccharification and co-fermentation (SSCF)

A 5 L bioreactor was used for handling the high solids loading SSCFof the pretreated and biodetoxified corn stover feedstock (Zhang et al.,2010b). The prehydrolysis was conducted at the solids loading of 30%(w/w) and the cellulase dosage of 12 FPU/g DM for 6 h at 50 °C and pH4.8. The SSCF started by inoculating the P. acidilactici strain (10%, v/v)at 42 °C for 72 h. The pH during SSCF was maintained at 5.5 by auto-matically feeding of 13.5 M NaOH solution or 6.75M Ca(OH)2 slurry.The samples were taken at regular intervals for HPLC and cell viabilityanalysis.

Fig. 1. Flowchart of cellulosic L-lactic acid production from corn stover feedstock on Aspen Plus platform.

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2.5. Analysis and calculation

Glucose, xylose, and lactic acid in the samples were analyzed onHPLC (LC-20AD pump, RI detector RID-10 A, Shimadzu, Kyoto, Japan)with a Bio-Rad Aminex HPX-87H column, which was operated at 65 °Cwith 0.6mL/min of 5mM H2SO4 as the mobile phase. The lactic acidyield of SSCF was calculated according to the method described by Qiuet al. (2017, 2018).

2.6. Process model establishment and techno-economic analysis

Process model was built up based on the NREL design for ethanolproduction (Humbird et al., 2011) and our previous established modelfor L-lactic acid production (Liu et al., 2015) from corn stover usingAspen Plus software (Aspen Tech, Cambridge, MA). The biorefiningplant size was 900 tons processing capacity of corn stover each day(300,000 tons annually) with an annual operation of 8000 h. Theoverall flowsheet of the present model in Fig. 1 consisted of 10 areas,including feedstock preprocessing (A100), pretreatment (A200), bio-detoxification (A250), hydrolysis and fermentation (A300), cellulasefermentation (A400), product recovery (A500), wastewater treatment

(A600), storage (A700), boiler (A800), and utilities (A900). The maindifferences between the previous and the current process models forcellulosic L-lactic acid production were pH neutralizing agents andproduct recovery methods, as follows:

(1) In Area A300, the neutralizing agents used were different. For theprevious model, liquid NaOH was used and sodium lactate wasformed; For the current model, Ca(OH)2 slurry was used and cal-cium lactate was formed.

(2) In Area A500, the purification process was different. For the pre-vious model, the purification process including solid/liquid se-paration, ultrafiltation, electrodialysis and multi-effect evaporationwas conducted successively to obtain 88% (w/w) lactic acid solu-tion. For the current model, the fermentation slurry experiencedsolid/liquid separation, acidification (sulfuric acid supplementa-tion), centrifugation, and multi-effect evaporation, then 88% (w/w)lactic acid solution was obtained.

Techno-economic analysis of cellulosic lactic acid production wasperformed under the assumption of “nth-plant” economics. The massand energy balance data (shown in Fig. A.1) from the Aspen Plus si-mulation were used to assist in determining the number and size ofequipment and chemical usage. All costs were indexed to 2013 with theexchange rate from dollar to Chinese Yuan of 1:6.2. The prices of pre-treatment reactors, fermentation tanks and helical agitators used werequoted from manufactures in China in 2014 (shown in Table A.1).Chemical prices were calculated according to the actual situations ofChina (shown in Table A.2). The total capital investment was calculatedbased on the total equipment costs. The variables and fixed operatingcosts were determined according to the plant capacity. The discountedcash flow rate of return (DCFROR) analysis to determine the minimumselling price (MSP, $/kg) of cellulosic lactic acidrequired to obtain a netpresent value of zero with 8% internal rate of return after taxes. Theparameters used in the DCFROR analysis were shown in Table A.3. Theraw material composition, main equipments and chemical used in theprocess referred to Liu et al. (2015).

3. Results and discussion

3.1. Improved L- and D-lactic acid yield by recovering the acids in calciumlactate form

The cellulosic lactic acid fermentation under the high solids loadingSSCF was evaluated by recovering the acids in the forms of either cal-cium or sodium lactates using Ca(OH)2 or NaOH as the neutralizingagents (Fig. 2). When L-lactic acid was produced from glucose andxylose release from corn stover feedstock by P. acidilactici ZY271(Fig. 2a), the final L-lactic acid titer and productivity were increased to130.3 ± 0.4 g/L and 1.81 ± 0.01 g/L/h, respectively, using Ca(OH)2as the neutralizing agent compared to those of 114.3 ± 0.9 g/L and1.59 ± 0.01 g/L/h, respectively using NaOH after 72 h’s SSCF(Table 1). The L-lactic acid yield was elevated from 74.7 ± 1.3% in thecase of NaOH use to 85.3 ± 0.3% in the case of Ca(OH)2 use. When D-lactic acid was produced by P. acidilactici ZY15 (Fig. 2b), the final D-lactic acid titer and productivity were 124.8 ± 1.1 g/L and1.73 ± 0.02 g/L/h, respectively using Ca(OH)2, comparing to112.2 ± 0.7 g/L and 1.56 ± 0.01 g/L/h, respectively using NaOH asthe neutralizing agent. The D-lactic acid yield was elevated from72.4 ± 0.7% of NaOH use to 80.7 ± 1.0% of Ca(OH)2 use.

It seems that xylose conversion was not affected (∼80%) by thedifferent neutralizing agents used and the different lactate salts formed(Table 1). But the amount of maximum viable cells in the case of NaOHuse was only two thirds of that in the case of Ca(OH)2 use, which mightbe responsible for the poor fermentation performance.

To our knowledge, L-lactic acid of 130.3 g/L with the yield of 85.3%(based on glucan and xylan in the raw corn stover feedstock) and D-

Fig. 2. Simultaneous saccharification and L-lactic acid co-fermentation (SSCF)using NaOH and Ca(OH)2 as the neutralizing agents. (a) L-lactic acid fermen-tation in sodium lactate or calcium lactate forms; (b) D-lactic acid fermentationin sodium lactate or calcium lactate forms. Prehydrolysis was conducted at50 °C and pH 4.8 for 6 h at cellulase dosage of 12 FPU/g DM. 10% (v/v) of the P.acidilactici inoculum, 42 °C for 72 h. P. acidilactici ZY271 and P. acidilactici ZY15were used for L-lactic acid and D-lactic acid fermentations.

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lactic acid of 124.8 g/L with the yield of 80.7% have been the highestcellulosic lactic acid fermentation levels reported among the relatedstudies.

3.2. Lactic acid in calcium lactate form reduced the inhibition on cellulaseactivity and cell viability

Although calcium lactate has a relatively low solubility (∼85 g/L at30 °C) than sodium lactate, and the calcium lactate concentration ob-tained is above its saturated solubility in water (> 120 g/L vs. 85 g/L),no calcium lactate precipitate was found during the whole SSCF processbecause of the complicated fermentation environment (mixing and highionic strength), and the strict requirements for crystallization of cal-cium lactate (Xu and Xu, 2014). But for 1M lactic acid produced, only0.5 M calcium lactate forms relative to 1M sodium lactate, whichmeans that less ionic strength or osmotic pressure provided by calciumlactate than sodium lactate.

On the other hand, cellulose conversion and fermenting cell viabi-lity are the two determinant factors on the cellulosic lactic acid yield.Thus, enzymatic hydrolysis of corn stover and lactic acid fermentationin the presence of different lactate salts were investigated. Fig. 3 showsthat the hydrolysis yield decreased with the increasing lactate con-centration. However, the higher inhibition was observed when thelactate was in sodium lactate form than in calcium lactate form at thesame lactate anions concentration. The partial reason was the lowerionic strength of the environment caused by the divalent cation of Ca2+

(Liu et al., 2014), which might be beneficial for the enzymatic hydro-lysis.

The cell viability of P. acidilactici ZY112 was also strongly affected

by the high lactate concentration (Fig. 4). The glucose consumption andlactic acid production almost ceased when sodium lactate was added toan equivalent lactic acid concentration of 80 g/L. In contrast, whencalcium lactate was added to the same lactic acid concentration, theglucose consumption and the lactic acid generation were not sig-nificantly affected (Fig. 4a). The cell viability of P. acidilactici TY112decreased significantly to 1–2×109 CFU/mL after the sodium lactateaddition, but the calcium lactate addition did not lead to a significantdecrease (6–7×109 CFU/mL) (Fig. 4b). Clearly, the lactic acid in thecalcium lactate form gave less osmotic stress on P. acidilactici cells thanin the sodium lactate form (Nakano et al., 2012). The high cell viability

Table 1L- and D-lactic acid fermentation using Ca(OH)2 and NaOH as the neutralizing agents for pH control.

Chirality Fermenting strain Neutralizing agent Maximum cell viability (CFU/mL) Titer (g/L) Yield (%) Productivity (g/L/h) Xylose conversion (%)

L-lactic acid P. acidilactici ZY271 NaOH 4.3 ± 0.1×109 114.3 ± 0.9 74.7 ± 1.3 1.59 ± 0.01 81.0 ± 0.0Ca(OH)2 6.2 ± 0.7×109 130.3 ± 0.4 85.3 ± 0.3 1.81 ± 0.01 83.0 ± 0.3

D-lactic acid P. acidilactici ZY15 NaOH 8.1 ± 1.7×109 112.2 ± 0.7 72.4 ± 0.7 1.56 ± 0.01 77.0 ± 5.2Ca(OH)2 13.9 ± 1.0× 109 124.8 ± 1.1 80.7 ± 1.0 1.73 ± 0.02 73.5 ± 0.8

Corn stover was pretreated, detoxified, and before the SSCF operations at 30% (w/w) solids loading. SSCF conditions were listed below Fig. 2.

Fig. 3. Enzymatic hydrolysis assay of the pretreated corn stover feedstock atvarying lactate concentrations. Corn stover was dry acid pretreated and bio-detoxified. Enzymatic hydrolysis was conducted at 12 FPU/g DM, 42 °C, pH 5.5for 12 h at 2.5% (w/w) solids loading. Calcium lactate or sodium lactate wasadded into the hydrolysates before enzymatic hydrolysis. The relative glucoseyield was calculated with the hydrolysis assay without lactate addition ascontrol.

Fig. 4. Lactic acid fermentation under existence of high sodium or calciumlactate concentrations. (a) L-lactic acid production. (b) Cell viability. The fer-mentation was conducted at 42 °C for 48 h in the simplified MRS medium.Sodium or calcium lactates were added to the simplified MRS medium to themolar concentration of 0.89mol/L of sodium lactate or 0.45mol/L of calciumlactate (equivalent to 80 g/L of lactic acid) at 3 h after the start of fermentation.L-lactic acid concentration was the lactic acid produced from the fermentationand the added L-lactate was not included.

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of the fermenting strains in the calcium lactate environment should beone of the major reasons for the better lactic acid fermentation per-formance.

Similar to the lactic acid production using starch feedstock (Liuet al., 2014), cellulosic lactic acid production by the high solids loadingSSCF also showed the better conversion yield when the lactic acid wasrecovered in the form of calcium lactate, instead of sodium lactate. Thelessened lactate inhibition on the cellulase activity and cell viabilitiesare the major reasons of the elevation of lactic acid yield. On the otherhand, the use of Ca(OH)2 slurry as the neutralizing agent easily pre-cipitates and then blocks the pipeline relative to the NaOH solution. Aproper feeding procedure of Ca(OH)2 slurry should be carefully con-sidered in the practical operation.

3.3. Techno-economic analysis of cellulosic lactic acid production

The techno-economic analysis was carried out based on Aspen Plusmodeling as described in Section 2.6. The lactic acid product refers tothe 88% (w/w) lactic acid solution. For the case of L-lactic acid pro-duction in sodium lactate form, one ton corn stover could produce0.384 ton lactic acid (Fig. A.1). The minimum selling price (MSP) of L-lactic acid is $0.529 per kg, in which the contributions of feedstock,enzyme, and non-enzyme conversion were $0.161, $0.082, and $0.287per kg, respectively (Table 2). The MSP of L-lactic acid obtain in thisstudy was relative lower compared with our previous results ($0.559per kg), which was caused by the relative higher lactic acid titer andyield (Liu et al., 2015). For the case of L-lactic acid production in cal-cium lactate form, 0.424 ton lactic acid was obtained using one ton cornstover. The MSP of L-lactic acid is $0.488 per kg, which is lower thanthat in sodium lactate form. For the D-lactic acid production, a similartendency was observed. That is, the yield of D-lactic acid in calciumlactate form (0.406 ton per ton corn stover) was higher than in sodiumlactate form (0.377 ton per ton corn stover). The MSP of D-lactic acidproduction in calcium lactate form ($0.509 per kg) is much lower thanthat in sodium lactate form ($0.539 per kg). The MSP of L-lactic acid isslightly lower relative to that of D-lactic acid. The higher lactic acidyield obtained in calcium lactate form compared with that in sodiumlactate form leads to the lower MSP. The calculated MSP of cellulosiclactic acid was almost one third of the market price of lactic acid(∼$1.3/kg, Alibaba Enterpriser, https://www.1688.com), which de-monstrated the great potential of lactic acid production using lig-nocellulosic feedstocks to replace the food crop materials.

4. Conclusion

Simultaneous saccharification and co-fermentation (SSCF) of thepretreated and biodetoxified corn stover increased 14.0% of L-lacticacid yield to 130.3 g/L in calcium lactate form using Ca(OH)2 as theneutralizing agent relative to sodium lactate form using NaOH forneutralization. Similarly, D-lactic acid yield was increased by 11.2% to

124.8 g/L in calcium lactate form relative to sodium lactate form. Thelessened lactate inhibition in calcium lactate form on the cellulase ac-tivity and the cell viability of the fermenting strains was found to beresponsible for the elevated cellulosic L- and D-lactic acid yield. Inaddition, greater economic advantage of chiral lactic acid production incalcium lactate than that in sodium lactate was demonstrated bytechno-economic analysis.

Acknowledgements

This research was supported by the National Key Research andDevelopment Program of China (2017YFB0309302) Shanghai PujiangProgram, the Fundamental Research Funds for the Central Universitiesof China (WF1514325, WF1814033), China Postdoctoral ScienceFoundation (2018M632043), and the Open Funding Project of the KeyLaboratory for Solid Waste Management and Environment Safety(SWMES2017-10), Ministry of Education of China, Tsinghua University(Beijing, China).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.indcrop.2018.10.041.

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Table 2Impact of different lactic acid salts produced on the minimum selling price(MSP) of cellulosic lactic acid calculated based on the Aspen Plus modelling.

MSP ($/kg) L-Sodiumlactate

L-Calciumlactate

D-Sodiumlactate

D-Calciumlactate

Lactic acid yield(%)

74.7 85.3 72.4 80.7

MSP ($/kg) 0.529 0.488 0.539 0.509Feedstock cost

($/kg)0.161 0.146 0.164 0.152

Enzyme cost($/kg)

0.082 0.074 0.083 0.078

Conversion cost($/kg)

0.287 0.268 0.292 0.279

The detailed SSCF conditions were listed below Fig. 2.

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